ATP-dependent proteases in biogenesis and maintenance of plant mitochondria

ATP-dependent proteases from three families have been identified experimentally in Arabidopsis mitochondria: four FtsH proteases (AtFtsH3, AtFtsH4, AtFtsH10, and AtFtsH11), two Lon proteases (AtLon1 and AtLon4), and one Clp protease (AtClpP2 with regulatory subunit AtClpX). In this review we discuss their submitochondrial localization, expression profiles and proposed functions, with special emphasis on their impact on plant growth and development. The best characterized plant mitochondrial ATP-dependent proteases are AtLon1 and AtFtsH4. It has been proposed that AtLon1 is necessary for proper mitochondrial biogenesis during seedling establishment, whereas AtFtsH4 is involved in maintaining mitochondrial homeostasis late in rosette development under short-day photoperiod.     

The significance of Arabidopsis AAA proteases for activity and assembly/stability of mitochondrial OXPHOS complexes

The Arabidopsis genome encodes four mitochondrially localized adenosine 5'-triphosphate-dependent metalloproteases called FtsH or AAA proteases. All of them span the inner mitochondrial membrane but the catalytic site of two of them (AtFtsH4 and AtFtsH11) faces the intermembrane space, while AtFtsH3 and AtFtsH10 face the matrix. We used a combination of blue-native polyacrylamide gel electrophoresis and histochemical staining to reveal the consequences of the loss of one of mitochondrial FtsHs on the efficiency of the oxidative phosphorylation system in Arabidopsis mitochondria. To address this issue, we have selected homozygous lines of respective transferred DNA (T-DNA) insertional mutants. A decrease in the activity of complexes I and V but not complex IV was observed in the ftsh mutants, except for the mutant lacking functional FtsH11. The reduced activity of complexes I and V was well correlated with a decreased protein level of these complexes. Western blots experiments using specific antibodies against complex V subunits showed a significant reduction of these subunits only in the ftsh4 mutant. Taken together, our results reveal a role of FtsH3, FtsH4 and FtsH10 proteases in the biogenesis of a plant oxidative phosphorylation system.     

The lack of mitochondrial AtFtsH4 protease alters Arabidopsis leaf morphology at the late stage of rosette development under short-day photoperiod

AtFtsH4 is one of four inner membrane-bound mitochondrial ATP-dependent metalloproteases in Arabidopsis thaliana , called AAA proteases, whose catalytic site is exposed to the intermembrane space. In the present study, we used a reverse-genetic approach to investigate the physiological role of the AtFtsH4 protease. We found that loss of AtFtsH4 did not significantly affect Arabidopsis growth under optimal conditions (long days); however, severe morphological and developmental abnormalities in late rosette development occurred under short-day conditions. The asymmetric shape and irregular serration of expanding leaf blades were the most striking features of the ftsh4 mutant phenotype. The severe abnormal morphology of the leaf blades was accompanied by ultrastructural changes in mitochondria and chloroplasts. These abnormalities correlated with elevated levels of reactive oxygen species and carbonylated mitochondrial proteins. We found that two classes of molecular chaperones, Hsp70 and prohibitins, were over-expressed in ftsh4 mutants during late vegetative growth under both short- and long-day conditions. Taken together, our data indicate that lack of AtFtsH4 results in impairment of organelle development and Arabidopsis leaf morphology under short-day conditions.     

Functional analysis of a recently originating,atypical presequence: mitochondrial import and processing of GUS fusion proteins in transgenic tobacco and yeast

A gene family of at least five members encodes the tobacco mitochondrial Rieske Fe-S protein (RISP). To determine whether all five RISPs are translocated to mitochondria, fusion proteins containing the putative presequences of tobacco RISPs and Escherichia coli -glucuronidase (GUS) were expressed in transgenic tobacco, and the resultant GUS proteins were localized by cell fractionation. The aminoterminal 75 and 71 residues of RISP2 and RISP3, respectively, directed GUS import into mitochondria, where fusion protein processing occurred. The amino-terminal sequence of RISP4, which contains an atypical mitochondrial presequence, can translocate the GUS protein specifically into tobacco mitochondria with apparently low efficiency.Consistent with the proposal of a conserved mechanism for protein import in plants and fungi, the tobacco RISP3 and RISP4 presequences can direct import and processing of a GUS fusion protein in yeast mitochondria. Plant presequences, however, direct mitochondrial import in yeast less efficiently than the yeast presequence, indicating subtle differences between the plant and yeast mitochondrial import machineries. Our studies show that import of RISP4 may not require positively charged amino acid residues and an amphipathic secondary structure; however, these structural properties may improve the efficiency of mitochondrial import.     

Inactivation of YME1, a member of the ftsH-SEC18-PAS1-CDC48 family of putative ATPase-encoding genes, causes increased escape of DNA from mitochondria in Saccharomyces cerevisiae.

The yeast nuclear gene YME1 was one of six genes recently identified in a screen for mutations that elevate the rate at which DNA escapes from mitochondria and migrates to the nucleus. yme1 mutations, including a deletion, cause four known recessive phenotypes: an elevation in the rate at which copies of TRP1 and ARS1, integrated into the mitochondrial genome, escape to the nucleus; a heat-sensitive respiratory-growth defect; a cold-sensitive growth defect on rich glucose medium; and synthetic lethality in rho- (cytoplasmic petite) cells. The cloned YME1 gene complements all of these phenotypes. The gene product, Yme1p, is immunologically detectable as an 82-kDa protein present in mitochondria. Yme1p is a member of a family of homologous putative ATPases, including Sec18p, Pas1p, Cdc48p, TBP-1, and the FtsH protein. Yme1p is most similar to the Escherichia coli FtsH protein, an essential protein involved in septum formation during cell division. This observation suggests the hypothesis that Yme1p may play a role in mitochondrial fusion and/or division.     

Functional redundancy of AtFtsH metalloproteases in thylakoid membrane complexes

FtsH is an ATP-dependent metalloprotease found in bacteria, mitochondria, and plastids. Arabidopsis (Arabidopsis thaliana) contains 12 AtFtsH proteins, three in the mitochondrion and nine in the chloroplast. Four of the chloroplast FtsH proteins are encoded by paired members of closely related genes (AtFtsH1 and 5, and AtFtsH2 and 8). We have previously reported that AtFtsH2 and 8 are interchangeable components of AtFtsH complexes in the thylakoid membrane. In this article, we show that the var1 variegation mutant, which is defective in AtFtsH5, has a coordinate reduction in the AtFtsH2 and 8 pair, and that the levels of both pairs are restored to normal in var1 plants that overexpress AtFtsH1. Overexpression of AtFtsH1, but not AtFtsH2/VAR2, normalizes the pattern of var1 variegation, restoring a nonvariegated phenotype. We conclude that AtFtsH proteins within a pair, but not between pairs, are interchangeable and functionally redundant, at least in part. We further propose that the abundance of each pair is matched with that of the other pair, with excess subunits being turned over. The variegation phenotype of var1 (as well as var2, which is defective in AtFtsH2) suggests that a threshold concentration of subunits is required for normal chloroplast function. AtFtsH1, 2, 5, and 8 do not show evidence of tissue or developmental specific expression. Phylogenetic analyses revealed that rice (Oryza sativa) and Arabidopsis share a conserved core of seven FtsH subunit genes, including the AtFtsH1 and 5 and AtFtsH2 and 8 pairs, and that the structure of the present-day gene families can be explained by duplication events in each species following the monocot/dicot divergence.     

Electron cryomicroscopy structure of a membrane-anchored mitochondrial AAA protease

FtsH-related AAA proteases are conserved membrane-anchored, ATP-dependent molecular machines, which mediate the processing and turnover of soluble and membrane-embedded proteins in eubacteria, mitochondria, and chloroplasts. Homo- and hetero-oligomeric proteolytic complexes exist, which are composed of homologous subunits harboring an ATPase domain of the AAA family and an H41 metallopeptidase domain. Mutations in subunits of mitochondrial m-AAA proteases have been associated with different neurodegenerative disorders in human, raising questions on the functional differences between homo- and hetero-oligomeric AAA proteases. Here, we have analyzed the hetero-oligomeric yeast m-AAA protease composed of homologous Yta10 and Yta12 subunits. We combined genetic and structural approaches to define the molecular determinants for oligomer assembly and to assess functional similarities between Yta10 and Yta12. We demonstrate that replacement of only two amino acid residues within the metallopeptidase domain of Yta12 allows its assembly into homo-oligomeric complexes. To provide a molecular explanation, we determined the 12 Å resolution structure of the intact yeast m-AAA protease with its transmembrane domains by electron cryomicroscopy (cryo-EM) and atomic structure fitting. The full-length m-AAA protease has a bipartite structure and is a hexamer in solution. We found that residues in Yta12, which facilitate homo-oligomerization when mutated, are located at the interface between neighboring protomers in the hexamer ring. Notably, the transmembrane and intermembrane space domains are separated from the main body, creating a passage on the matrix side, which is wide enough to accommodate unfolded but not folded polypeptides. These results suggest a mechanism regarding how proteins are recognized and degraded by m-AAA proteases.     

<Emphasis Type="Italic">GmFtsH9</Emphasis> expression correlates with in vivo photosystem II function: chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence transient analysis and eQTL mapping in soybean

Filamentation temperature-sensitive H (FtsH) is an ATP-dependent zinc metalloprotease involved in diverse biological functions. There are 12 FtsH proteins in Arabidopsis, among which AtFtsH2 plays an important role in regulating the turnover of photosystem II (PSII) reaction center D1 protein and the development of the photosynthetic apparatus. Here, we have identified 11 FtsH genes in the soybean genome by a bioinformatics approach. These soybean FtsH genes corresponded to seven Arabidopsis FtsH genes, suggesting that the main characteristics of soybean FtsH genes were formed before the evolutionary split of soybean and Arabidopsis. Phylogenetic analyses allowed us to clone a soybean AtFtsH2-like gene designated as GmFtsH9. The predicted protein of GmFtsH9 consists of 690 amino acids and contains three typical FtsH proteins conserved domains. The expression level of GmFtsH9 was determined in a soybean recombinant inbred line population under a pot experiment conducted for measuring chlorophyll a fluorescence transient parameters, photosynthetic CO₂ fixation rate (P _N), and seed yield. Expression quantitative trait loci (eQTL) mapping revealed two trans-acting eQTLs for GmFtsH9. The significant correlation of gene expression level with chlorophyll a fluorescence transient parameters and the presence of overlapping eQTL (QTL) between gene expression level and chlorophyll a fluorescence transient parameters indicated that GmFtsH9 could be involved in regulating PSII function. These results further lead to the understanding of the mechanism underlying FtsH gene expression, and contribute to the development of marker-assisted selection breeding programs for modulating soybean FtsH gene expression.     

Role of the AAA protease Yme1 in folding of proteins in the intermembrane space of mitochondria

The vast majority of mitochondrial proteins are synthesized in the cytosol and transported into the organelle in a largely, if not completely, unfolded state. The proper function of mitochondria thus depends on folding of several hundreds of proteins in the various subcompartments of the organelle. Whereas folding of proteins in the mitochondrial matrix is supported by members of several chaperone families, very little is known about folding of proteins in the intermembrane space (IMS). We targeted dihydrofolate reductase (DHFR) as a model substrate to the IMS of yeast mitochondria and analyzed its folding. DHFR can fold in this compartment, and its aggregation upon heat shock can be prevented in an ATP-dependent manner. Yme1, an AAA (ATPases associated with diverse cellular activities) protease of the IMS, prevented aggregation of DHFR. Analysis of protein aggregates in mitochondria lacking Yme1 revealed the presence of a number of proteins involved in the establishment of mitochondrial ultrastructure, lipid metabolism, protein import, and respiratory growth. These findings explain the pleiotropic effects of deletion of YME1 and suggest an important role for Yme1 as a folding assistant, in addition to its proteolytic function, in the protein homeostasis of mitochondria     

In vitro and in vivo studies on the mitochondrial import of CBS1, a translational activator of cytochrome b in yeast

Summary Translation of mitochondrial cytochrome b mRNA in yeast is activated by the product of the nuclear gene CBS1. CBS1 encodes a 27 kDa precursor protein, which is cleaved to a 24 kDa mature protein during the import into isolated mitochondria. The sequences required for mitochondrial import reside in the amino-terminal end of the CBS1 precursor. Deletion of the 76 amino-terminal amino acids renders the protein incompetent for mitochondrial import in vitro and non-functional in vivo. When present on a high copy number plasmid and under the control of a strong yeast promoter, biological function can be restored by this truncated derivative. This observation indicates that the CBS1 protein devoid of mitochondrial targeting sequences can enter mitochondria in vivo, possibly due to a bypass of the mitochondrial import system.     

Biochemical and functional analysis of the YME1 gene product, an ATP and zinc-dependent mitochondrial protease from S. cerevisiae.

Inactivation of YME1 in yeast causes several distinct phenotypes: an increased rate of DNA escape from mitochondria, temperature-sensitive growth on nonfermentable carbon sources, extremely slow growth when mitochondrial DNA is completely absent from the cell, and altered morphology of the mitochondrial compartment. The protein encoded by YME1, Yme1p, contains two highly conserved sequence elements, one implicated in the binding and hydrolysis of ATP, and the second characteristic of active site residues found in neutral, zinc-dependent proteases. Both the putative ATPase and zinc-dependent protease elements are necessary for the function of Yme1p as genes having mutations in critical residues of either of these motifs are unable to suppress any of the phenotypes exhibited by yme1 deletion strains. Yme1p co-fractionates with proteins associated with the mitochondrial inner membrane, is tightly associated with this membrane, and is oriented with the bulk of the protein facing the matrix. Unassembled subunit II of cytochrome oxidase is stabilized in yme1 yeast strains. The data support a model in which Yme1p is an ATP and zinc-dependent protease associated with the matrix side of the inner mitochondrial membrane. Subunit II of cytochrome oxidase, when not assembled into a higher order complex, is a likely substrate of Yme1p.     

Immunologically related proteins in cytoplasmic and mitochondrial ribosomes of yeast Saccharomyces cerevisiae

Summary Ribosomal proteins from the cytoplasm and mitochondria of the yeast Saccharomyces cerevisiae were compared by immunoblotting techniques. Antibodies raised against cytoplasmic ribosomal proteins cross-react with five mitochondrial ribosomal proteins, four of which are located in the large and one in the small mitochondrial subunits. The possible existence of common ribosomal proteins for cytoplasmic and mitochondrial ribosomes is discussed.Abbreviations cyto cytoplasmic - mito mitochondrial     

Autocatalytic Processing of m-AAA Protease Subunits in Mitochondria

m-AAA proteases are ATP-dependent proteolytic machines in the inner membrane of mitochondria which are crucial for the maintenance of mitochondrial activities. Conserved nuclear-encoded subunits, termed paraplegin, Afg3l1, and Afg3l2, form various isoenzymes differing in their subunit composition in mammalian mitochondria. Mutations in different m-AAA protease subunits are associated with distinct neuronal disorders in human. However, the biogenesis of m-AAA protease complexes or of individual subunits is only poorly understood. Here, we have examined the processing of nuclear-encoded m-AAA protease subunits upon import into mitochondria and demonstrate autocatalytic processing of Afg3l1 and Afg3l2. The mitochondrial processing peptidase MPP generates an intermediate form of Afg3l2 that is matured autocatalytically. Afg3l1 or Afg3l2 are also required for maturation of newly imported paraplegin subunits after their cleavage by MPP. Our results establish that mammalian m-AAA proteases can act as processing enzymes in vivo and reveal overlapping activities of Afg3l1 and Afg3l2. These findings might be of relevance for the pathogenesis of neurodegenerative disorders associated with mutations in different m-AAA protease subunits.     

Isolation of a cDNA encoding mitochondrial citrate synthase from Arabidopsis thaliana

We isolated a cDNA clone from Arabidopsis thaliana encoding the TCA cycle enzyme, citrate synthase. The plant enzyme displays 48% and 44% amino acid residue similarity with the pig, and yeast polypeptides, respectively. Many proteins, including citrate synthase, which are destined to reside in organelles such as mitochondria and chloroplasts, are the products of the nucleocytoplasmic protein synthesizing machinery and are imported post-translationally to the site of function. We present preliminary investigations toward the establishment of an in vitro plant mitochondrial import system allowing for future studies to dissect this process in plants where the cell must differentiate between mitochondria and chloroplast and direct their polypeptides appropriately.     

A new Escherichia coli gene, fdrA, identified by suppression analysis of dominant negative FtsH mutations

An Escherichia coli membrane protein, FtsH, has been implicated in several cellular processes, including integration of membrane proteins, translocation of secreted proteins, and degradation of some unstable proteins. However, how it takes part in such diverse cellular events is largely unknown. We previously isolated dominant negative ftsH mutations and proposed that FtsH functions in association with some other cellular factor(s). To test this proposal we isolated multicopy suppressors of dominant negative ftsH mutations. One of the multicopy suppressor clones contained an N-terminally truncated version of a new gene that was designated fdrA. The FdrA fragment suppressed both of the phenotypes — increased abnormal translocation of a normally cytoplasmic domain of a model membrane protein and retardation of protein export — caused by dominant negative FtsH proteins. The intact fdrA gene (11.9 min on the chromosome) directed the synthesis of a 60 kDa protein in vitro.     

Inorganic polyphosphates and exopolyphosphatases in different cell compartments of Saccharomyces cerevisiae

The cytosol, nuclei, vacuoles, and mitochondria of the yeast Saccharomyces cerevisiae possess inorganic polyphosphates (polyPs). PolyP levels, spectra of polyP chain lengths, and their dependence on the growth phase are distinguished in the mentioned compartments. Inactivation of the PPX1 gene has no effect on the polyP metabolism under cultivation of the yeast in medium with glucose and 5–7 mM P_i. Inactivation of the PPN1 gene results in elimination of the high-molecular-mass exopolyphosphatases (∼120 to 830 kD) of the cytosol, nuclei, vacuoles, and mitochondria of S. cerevisiae suggesting that it is just PPN1 that encodes these enzymes. Expression of the low-molecular-mass exopolyphosphatase of ∼45 kD encoded by the PPX1 gene decreases under PPN1 inactivation as well. While PPN1 inactivation has negligible effect on polyP levels, it results in increase in the long-chain polyPs in all the compartments under study. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 11, pp. 1445–1450.     

Deletion of FtsH11 protease has impact on chloroplast structure and function in Arabidopsis thaliana when grown under continuous light

The membrane‐integrated metalloprotease FtsH11 of Arabidopsis thaliana is proposed to be dual‐targeted to mitochondria and chloroplasts. A bleached phenotype was observed in ftsh11 grown at long days or continuous light, pointing to disturbances in the chloroplast. Within the chloroplast, FtsH11 was found to be located exclusively in the envelope. Two chloroplast‐located proteins of unknown function (Tic22‐like protein and YGGT‐A) showed significantly higher abundance in envelope membranes and intact chloroplasts of ftsh11 and therefore qualify as potential substrates for the FtsH11 protease. No proteomic changes were observed in the mitochondria of 6‐week‐old ftsh11 compared with wild type, and FtsH11 was not immunodetected in these organelles. The abundance of plastidic proteins, especially of photosynthetic proteins, was altered even during standard growth conditions in total leaves of ftsh11. At continuous light, the amount of photosystem I decreased relative to photosystem II, accompanied by a drastic change of the chloroplast morphology and a drop of non‐photochemical quenching. FtsH11 is crucial for chloroplast structure and function during growth in prolonged photoperiod.     

Proteolytic processing of Atg32 by the mitochondrial i-AAA protease Yme1 regulates mitophagy

Mitophagy, the autophagic removal of mitochondria, occurs through a highly selective mechanism. In the yeast Saccharomyces cerevisiae, the mitochondrial outer membrane protein Atg32 confers selectivity for mitochondria sequestration as a cargo by the autophagic machinery through its interaction with Atg11, a scaffold protein for selective types of autophagy. The activity of mitophagy in vivo must be tightly regulated considering that mitochondria are essential organelles that produce most of the cellular energy, but also generate reactive oxygen species that can be harmful to cell physiology. We found that Atg32 was proteolytically processed at its C terminus upon mitophagy induction. Adding an epitope tag to the C terminus of Atg32 interfered with its processing and caused a mitophagy defect, suggesting the processing is required for efficient mitophagy. Furthermore, we determined that the mitochondrial i-AAA protease Yme1 mediated Atg32 processing and was required for mitophagy. Finally, we found that the interaction between Atg32 and Atg11 was significantly weakened in yme1∆ cells. We propose that the processing of Atg32 by Yme1 acts as an important regulatory mechanism of cellular mitophagy activity.